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NITROGEN AND PHOSPHORUS UPTAKE BY CULTURED MICROBES COLLECTED FROM THE LAS VEGAS WASH AND ASSOCIATED AREAS Prepared by: Henry Sun, Ph.D Division of Earth and Ecosystem Sciences Desert Research Institute Las Vegas, Nevada 89119-7363 September 2010

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Page 1: NITROGEN AND PHOSPHORUS UPTAKE BY CULTURED MICROBES ... · Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 13 study alga

NITROGEN AND PHOSPHORUS UPTAKE BY CULTURED MICROBES COLLECTED FROM THE LAS VEGAS WASH

AND ASSOCIATED AREAS

Prepared by:

Henry Sun, Ph.D Division of Earth and Ecosystem Sciences

Desert Research Institute Las Vegas, Nevada 89119-7363

September 2010

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas i

ABSTRACT

In Las Vegas, effluents from wastewater treatment plants are discharged into Lake Mead via Las Vegas Wash (Wash). Nutrients contained in these effluents, especially nitrate and phosphate, are a concern because they could promote algal growth in Lake Mead, the region’s primary water supply. To minimize this potential problem, several constructed wetlands have been established in various segments of the Wash as part of an erosion control program. Although these facilities are a consequence of stabilizing the stream channel, there is an expectation that that plants will grow and take up some of the nutrients. A recent report indicates that wetland plants indeed have a positive effect on water quality. The objective of this study is to determine whether algae play a similar role. Surveys indicate that in shallow parts of the Wash macroscopic algae grow to a considerable density in the summer months. In the laboratory, four cultures of microalgae, with one exception, were found to take up nitrate and phosphate from Wash water. Three bacteria isolated from the Wash, as expected, produce nitrate. These results indicate that algae do take up nutrients from the Wash, but the net benefit of this activity cannot be realized unless algal growth is removed from the system. Without removal, as is now the case, algae would ultimately die and decay, releasing the absorbed nutrients back to the Wash.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas ii

ACKNOWLEDGEMENTS I thank the Southern Nevada Water Authority for funding this study with a grant provided to them by the Bureau of Reclamation. I also thank my student Ms. Faegheh Moazeni for technical assistance, and Jim Pollard, Peggy Roefer, Todd Tietjen, Seth Shanahan, and Xiaoping Zhou for comments that improved the report.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas iii

Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas

Table of Contents

Page No.

Abstract ...................................................................................................................................... i Acknowledgements ................................................................................................................. ii Table of Contents ................................................................................................................... iii List of Tables .......................................................................................................................... iii List of Figures ..........................................................................................................................iv 1.0 INTRODUCTION ............................................................................................................ 1 2.0 METHODS ........................................................................................................................ 2 3.0 RESULTS AND DISCUSSION ..................................................................................... 4 3.1 Algae Survey ............................................................................................................ 4 3.2 Algae Cultured ........................................................................................................ 8 3.3 Nitrate Bioavailability ............................................................................................ 9 3.4 Phosphate Bioavailability .................................................................................... 12 4.0 RECOMMENDATIONS .............................................................................................. 13 5.0 LITERATURE CITED ................................................................................................. 14

List of Tables Table 1. Compositions of algal growth media used in the study .................................. 2

Table 2. Observed benthic algae represented in Las Vegas Wash at Pabco Road Weir

and an estimate of their relative abundance ...................................................... 6

Table 3. Observed algae represented at site 10.75 and an estimate of their relative

abundance............................................................................................................ 8

Table 4. Algae cultured from Las Vegas Wash and associated wetlands. These were

used to assess nitrate and phosphate availability of Las Vegas Wash water. . 8

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas iv

Page No.

List of Figures Figure 1. Map showing study locations (black dots) along Flamingo Wash and Las

Vegas Wash............................................................................................................ 3

Figure 2. Calibration curve for nitrate-N assay ................................................................... 3

Figure 3. Light micrograph of Cladophora sp. from Flamingo Wash. Filament is about

8 microns wide ....................................................................................................... 4

Figure 4. Top: Cladophora sp. bloom in Flamingo Wash. Bottom: quantifying algal

growth rate ............................................................................................................. 5

Figure 5. Las Vegas Wash at Pabco Road Weir, showing benthic algae growing on

concrete bottom near shore (green arrow) and on weir (red arrow) ................... 6

Figure 6. Scanning electron micrograph of diatom Stephanodiscus sp. from Pabco Road

Weir shown in Figure 5 ......................................................................................... 7

Figure 7. Las Vegas Wash at site 10.75. A mixture of diatoms and green algae grow on

concrete bottom...................................................................................................... 7

Figure 8. Light micrographs of Chlorella sp. (left) and of an unidentified diatom (right)

cultured from Las Vegas Wash and associated wetlands. Chlorella cells are

about 5 microns in diameter. Diatom filament is 8 microns wide ..................... 9

Figure 9. Wash water nitrate dynamics in presence of diatoms ....................................... 10

Figure 10. Wash water nitrate dynamics in presence of cyanobacterium

Oscillatoria sp ...................................................................................................... 11

Figure 11. Wash water nitrate dynamics in presence of bacteria ....................................... 11

Figure 12. Algal growth rate as a function of phosphate supplement in Wash water ....... 13

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 1

1.0 INTRODUCTION Algae are primitive photosynthetic organisms that grow in all aquatic habitats (van den Hoek et al., 1995). Ecologically, they play the same essential role as plants do on land. Simply put, they are the basis of the food chain that sustains all animal life. As such, they are a critical component of any healthy aquatic ecosystem. However, algal overgrowth (i.e. algal blooms), could cause environmental problems. At minimum, algal blooms are a nuisance. Biomass decay can quickly deplete oxygen in the hypolimnion, killing fish and other oxygen-requiring aquatic life (Diego-McGlone et al., 2008; Mhlanga et al., 2006). Worse, some algal blooms are toxic. For example, Microcystis, a single-celled freshwater cyanobacterium, produces microcystin, a potent heptatoxin (Nishizawa et al., 2001). Algal blooms in Lake Mead are of particular concern. The reservoir is not only used for recreational activities, it is also a source of drinking water for Las Vegas and downstream users. Currently, the concern is being managed through Total Maximum Daily Load restrictions for phosphorus in discharges from wastewater treatment facilities. These effluents flow into the Las Vegas Wash (hereafter, “Wash”) and, ultimately, into Lake Mead. The rationale is to withhold a critical nutrient from algae, thereby preventing blooms. Despite this control effort, a major algal bloom occurred in parts of the lake (Boulder Basin and Virgin Basin) in the spring of 2001. Fortunately, that bloom was caused primarily by a nontoxic alga, Pyramichlamys disecta. Cylindrospermopsis, a cyanobacterium that produces the toxin cylindrospermopsin, was also present, but only in small numbers. The 2001 incident indicates that Lake Mead was not entirely safe from algal blooms. Consequently, the wastewater dischargers have since been voluntarily removing phosphorus well beyond regulatory requirements. The fact that Lake Mead has not had any algal blooms in the intervening years may be attributable, at least in part, to the stringent control for phosphate. Since 1998, many erosion control structures (hereafter, “weir”) have been built in the Wash to reduce erosion. As a result, wetlands have been established behind each weir. Constructed wetlands are widely considered natural biological filtering systems that degrade or remove organic pollutants (de-Bashan and Bashan 2004). They are also expected to remove inorganic nutrients, such as phosphorus. Therefore, it is reasonable to hypothesize that similarly beneficial functions are performed by the wetlands associated with the Wash. Indeed, these constructed wetlands have by now developed into fully functional ecosystems, consisting of a host of aquatic plants and animal species. Research indicates that wetland plants have a positive influence on water quality (Acharya and Adhikari, 2010). The objective of this project is to investigate whether algae that live in the Wash and associated wetlands have a similar positive influence on water quality. This study will focus on the extent that the algae could absorb nitrate and phosphorus. Heterotrophic bacteria were also included in the study. Bacterial activity may negate the benefit from

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 2

the plants and algae, because decomposition could release the absorbed nutrients back into the Wash. 2.0 METHODS. From September to December 2009, monthly visits were made to the Flamingo Wash near the Desert Research Institute campus, the Wash at Pabco Road Weir, and site 10.75 just below Vegas Valley Drive (Figure 1). At each location, benthic and water samples were collected as grab samples and transported immediately to the laboratory for processing. In the laboratory, they were examined under a microscope, quantified for algal biomass, and cultured on BG-11 and diatom medium (Table 1). Algae that Table 1. Compositions of algal growth media used in the study. BG-11 Medium NaNO3 ……………………… 150 g/L K2HPO4…………………….. 4.0 g/L MgSO4.7H2O………………. 0.75 g/L CaCl2.2H2O………………… 3.6 g/L Citric acid.H2O…………….. 0.6 g/L Ammonium Ferric………….. 0.6 g/L Citrate Na2EDTA.2H2O…………… 0.1 g/L Na2CO3 …………………….. 2.0 g/L H3BO3……………………..... 2.86 g/L MnCl2.4H2O………………... 1.81 g/L ZnSO4.7H2O………………... 0.22 g/L Na2MoO4.2H2O…………….. 0.39 g/L CuSO4.5H2O………………... 0.079 g/L Co(NO3)2.6H2O…………….. 0.0494 g/L Diatom Medium Ca(NO3)2.4H2O…………….. 20.0 g/L KH2PO4……………………. 12.40 g/L MgSO4.7H2……………….... 25.0 g/L NaHCO3……………………. 15.9 g/L EDTAFeNa………………… 2.25 g/L EDTANa2 ………………….. 2.25 g/L H3BO3 ……………………... 2.48 g/L MnCl2.4H2O……………….. 1.39 g/L (NH4)6Mo7O24.4H2O………. 1.00 g/L Cyanocobalamin…………… 0.04 g/L Thiamine HCl……………… 0.04 g/L Biotin ……………………… 0.04 g/L NaSiO3.9H2O……………… 57 g/L Adjust to pH 6.9 with 1M HCl

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 3

Nitrate-N, mg/L

0 5 10 15 20 25

Abso

rban

ce a

t 423

nm

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

r² = 0.9951

Figure 1. Map showing study locations (black dots) along Flamingo Wash and Las Vegas Wash.

appeared in our cultures were purified to the extent that they were free of bacteria. A few bacteria were also isolated into pure culture. The uptake of nitrate and phosphate from Wash water by these organisms was determined. Algae in water samples were enumerated under a light microscope using a hemocytometer. Sixteen grids were counted, and the average cell count was calculated. In Flamingo Wash, where the alga was macroscopic, growth from a measured area was collected, air-dried, and weighed. Nitrate-N was determined by the use of the Hach Nitraver® 5 Nitrate Reagent. The content of one kit was dissolved in 10 mL of Nanopure® water. One milliliter (mL) of the reagent solution was combined with one mL of water sample. After letting it stand for ten minutes, its absorbance at the wavelength of 423 nm was determined in a spectrophotometer. As shown in Figure 2, the relationship between nitrate-N and absorbance is linear in the range of nitrate concentrations from 0 to 20 mg N per Liter.

Figure 2. Calibration curve for nitrate-N assay.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 4

3.0 RESULTS AND DISCUSSION 3.1 Algae Survey Flamingo Wash. In September, 2010, this tributary was taken over by the macroscopic green alga Cladophora sp. (Figure 3). This organism is cosmopolitan. Its dominance in Flamingo Wash may be due to a combination of slow flow and shallow water depth.

Figure 3. Light micrograph of Cladophora sp. from Flamingo Wash. Filament is about 8 microns wide.

To evaluate standing biomass of Cladophora sp., material was harvested from an area of 9.29 m2 (100 ft2) of the Wash (Figure 4). This yielded a total of 9 kg (20 lb) dry algal biomass. The cleared area appeared to recover to its previous extent in two weeks. This results in an anecdotal growth rate of about 69 grams (2.4 ounces) of dry weight per square meter per day. Las Vegas Wash. The number of algae in the center of the flow was below detection. No algae were found even after the sample was concentrated one thousand times by centrifugation. Thus, planktonic algae, if present, are below one cell per liter. Benthic algae were observed on the edge of the Wash where the water was stagnant (Figure 5, green arrow). Thick films of filamentous diatoms were also observed on the Pabco Road Weir (Figure 5, red arrow). The algae observed from these habitats are listed in Table 2. The dominant diatom species on the weir is Stephanodiscus sp. A scanning electron micrograph of this organism is shown in Figure 6.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 5

Figure 4. Top: Cladophora sp. bloom in Flamingo Wash. Bottom: quantifying algal growth rate.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 6

Table 2. Observed benthic algae represented in Las Vegas Wash at Pabco Road Weir and an estimate of their relative abundance.

Organisms Abundance*

Green Algae Chlorella sp. +

Dictyococcus varians + Rhizoclonium hieroglyphicum ++

Spirogyra sp. +++ Cyanobacteria Oscillatoria sp. +++ Diatoms Gomphonema sp. ++ Stephanodiscus sp. +++

*Note: +++ dominant, ++ intermediate abundance, + relative rare

Figure 5. Las Vegas Wash at Pabco Road Weir, showing benthic algae growing on concrete bottom near shore (green arrow) and on weir (red arrow).

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 7

Figure 6. Scanning electron micrograph of diatom Stephanodiscus sp. from Pabco Road Weir shown in Figure 5.

LW10.75. Water here, like water further downstream, is essentially free of planktonic algae. Algae grow mainly on the concrete bottom of the Wash in the form of benthic biofilm. Microscopic examination revealed a mixture of single celled green algae and diatoms (Figure 7 and Table 3).

Figure 7. Las Vegas Wash at site 10.75. A mixture of diatoms and green algae grow on concrete bottom.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 8

Table 3. Observed algae represented at site 10.75 and an estimate of their relative abundance.

Organisms Abundance*

Cyanobacteria Chlorogloea epiphytica ++ Chroococcus turgidus ++ Merismopedia glauca +++ Oscillatoria sp. + Diatoms Fragilaria crotonensis +

Gomphonema sp. + Unidentified diatom +

*Note: +++ dominant, ++ intermediate abundance, + relative rare 3.2 Algae Cultured Only some of the algae observed in the study sites could be cultured (Figure 8). These, listed below (Table 4), were used in nutrient uptake experiments. Table 4. Algae cultured from the Wash and associated wetlands. These were used to assess nitrate and phosphate availability of Las Vegas Wash water.

Green Algae

Chlorella sp. Cyanobacteria Oscillatoria sp. Diatoms Fragilaria crotonensis Gomphonema sp. Unidentified diatom

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 9

Figure 8. Light micrographs of Chlorella sp. (left) and of an unidentified diatom (right) cultured from Las Vegas Wash and associated wetlands. Chlorella cells are about 5 microns in diameter. Diatom filament is 8 microns wide.

3.3 Nitrate Bioavailability Four of the algae listed in Table 4 were studied to see if they can take up nitrate from Wash water. Three bacteria were studied to see if they produce nitrate by oxidizing ammonia available in Wash water. Briefly, the study organism was raised in appropriate medium. The population was collected by centrifugation, washed in sterile Wash water, and suspended in sterile Wash water. Wash water, collected at the Pabco Road Weir, was sterilized either by filtering or autoclaving. The latter resulted in a slight loss of nitrate-N. The culture was incubated with continuous shaking. For algae, illumination was provided with fluorescent lights at an intensity of about 1,000 lux or 13.5 mole m-2 s-2 (daily solar radiation maximum in Las Vegas is about 75,000 lux). The bacteria were incubated in the dark. At regular time intervals, the cultures were sampled. The samples were centrifuged to remove cells and particulates. The amount of nitrate-N remaining in Wash water was determined. The results for the algae experiments are shown in Figure 9. In the presence of the algae the amount of nitrate-N in the Wash water medium decreased. In the case of Gomphonema sp., it decreased from 10 mg/L to 1 mg/L. In the case of the unidentified diatom and Fragilaria crotonensis, nitrate-N decreased from 18 mg/L to 7 mg/L (ppm). After reaching these values, no further decline was observed. The absorbed nitrate may or may not stay absorbed. In diatom Fragilaria crotonensis, the level of nitrate-N stayed at 7 mg/L (ppm). In diatom Gomphonema sp. and the unidentified diatom, further incubation resulted in nitrate-N release, almost restoring its concentration to the initial level. This is presumably due to cell death and lysis, something that inevitably occurs to algae whether they grow in culture or nature. The result for the experiment with the cyanobacterium Oscillatoria sp. is shown in Figure 10. This result is similar to those for Gomphonema sp. and for the unidentified diatom. Initially, there was rapid nitrate uptake. This was followed by slow, gradual release of nitrate.

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Gomphonema sp.

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28 32 36 40 44 48

Nitr

ate-

N, m

g/L

0

2

4

6

8

10

12

(1.2*108 cells/ mL)

Unidentified diatom

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68

Nitr

ate-

N, m

g/L

4

6

8

10

12

14

16

18

20(1.17*105 cells/ mL)

Fragilaria crotonensis

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68

Nitr

ate-

N, m

g/L

6

8

10

12

14

16

18

20

(6.8*104 cells/ mL)

Figure 9. Wash water nitrate dynamics in presence of diatoms.

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 11

Oscillatoria sp.3.23x106 cells/mL

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28 32 36 40 44 48

Nitr

ate-

N, m

g/L

2

4

6

8

10

12

14

Figure 10. Wash water nitrate dynamics in presence of cyanobacterium Oscillatoria sp.

The bacteria were studied to determine how their activities would change nitrate concentrations in Wash water. The results for those experiments are shown in Figure 11. In the cultures that contained bacterium #1 and #2, the concentration of nitrate increased, from 14 mg/L to around 45 mg/L. This is probably evidence for nitrification activities. Presumably, the bacteria take up organic nitrogen, deaminate it (removing the amine group), and oxidize the carbon skeleton for energy. Ammonia, the product of deamination, is oxidized to nitrate. Nitrate in the culture of bacterium #3 decreased, not increased. This is not entirely unexpected. The range of organic compounds utilized by bacteria can vary widely from species to species. Bacterium #3 may be growing on carbohydrates. Because carbohydrates do not contain nitrogen, nitrate would be taken up as a nitrogen source.

Bacterium #1, Pabco

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28 32 36 40 44 48

Nitr

ate-

N, m

g/L

10

15

20

25

30

35

40

45

501.32*109 cells/ mL

Figure 11. Wash water nitrate dynamics in presence of bacteria (continued on page 12).

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Nitrogen and Phosphorus Uptake by Cultured Microbes Collected from the Las Vegas Wash and Associated Areas 12

Bacterium #2, Flamingo Wash

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28

Nitr

ate-

N, m

g/L

10

15

20

25

30

35

40

45

501.08*109 cells/ mL

Bacterium #3, Pabco

Time elapsed after inoculation, h

0 4 8 12 16 20 24 28 32 36 40 44 48

Nitr

ate-

N, m

g/L

2

4

6

8

10

12

14

16

18

20 1.2*109 cells/ mL

Figure 11. Wash water nitrate dynamics in presence of bacteria.

3.4 Phosphate Bioavailability The fact that algae grow in the Wash suggests the presence of algae-available phosphate. However, an uptake study similar to those done with nitrate is technically more challenging. Because there were limited resources available to complete this study, there were no routine phosphate assays that were of sufficient resolution (sub-ppm). To determine if the phosphate level is limiting, we took the green algal isolate Chlorella sp. and grew it in Wash water supplemented with various amounts of phosphate. The results are shown in Figure 12. It is evident that the amount of phosphate in Wash water is growth-limiting. With increases in available phosphate, the specific growth rate steadily increased until reaching a saturating concentration at 20 ppm. To the extent the

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study alga is representative of those in Lake Mead, this result suggests that the emission standard for wastewater treatment facilities with respect to phosphate in their effluents is a sound strategy for preventing algal blooms in Lake Mead.

Chlorella sp.

Amount of added phosphate (phosphate-P, mg/L)

0 10 20 30 40 502

4

6

8

10

12

Cel

l den

sity

, 10^

7 ce

lls/ m

L

Figure 12. Algal growth rate as a function of phosphate supplement in Wash water.

4.0 RECOMMENDATIONS Given the limited scope and preliminary nature of the study, caution should be exercised in basing management policy decisions on the data presented here. Nevertheless, some general conclusions are warranted. First, algae growing in the Wash and associated wetlands apparently have the potential to influence water quality. By absorbing the nutrients upstream, they could potentially reduce nutrient flux to Lake Mead. However, this benefit is realized only under certain conditions. For instance, the shallow, calm stream in the Flamingo Wash is ideal for abundant algal growth. In contrast, the deep, fast flow of the Wash is not suitable for algae except in localized niches. We don’t know the factors responsible for the general absence of algae in the Wash. Second, even in the Flamingo Wash, algae may not have a net benefit unless the biomass is removed from the system. Loss of nitrate-N can occur through denitrification activity. But phosphorus has no volatile forms. The only way for it to leave the system is physical removal. If, as currently is the case, algae are left in the Wash, they ultimately decompose, releasing the absorbed nutrients back to the environment. The situation is similar to wetland plants. If not harvested, they would ultimately decay, becoming a source of nutrients. There are several ways to create incentives for harvesting algae from the Flamingo Wash and possibly from the Wash associated wetlands. Algal biomass is a material in demand for biofuel production (Demirbas, 2008). Future studies should look

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into the biofuel potential of these algae. Another way to utilize algae is as organic fertilizers for horticulture and agriculture. Soils fertilized with composts are less susceptible to leaching than soils fertilized with chemicals (Steiner et al., 2008), which would reduce nutrient input to Lake Mead from non-point sources. Such waste-recycling practices could contribute to a sustainable economy in southern Nevada if they were conducted at a substantial scale. 5.0 LITERATURE CITED. Acharya, K., Adhikari A., 2010. A comparison of water quality improvements from three

different wetland types in the Las Vegas Valley watershed. Desert Research Institute, Las Vegas, NV.

Demirbas, A., 2008. Recent progress in biorenewable feedstocks. Energy Education Science and Technology, 22, 69-95.

de-Bashan, L.E., Bashan, Y., 2004. Recent advances in removing phosphorus from wastewater and its future use as fertilizer (1997-2003). Water Research, 38, 4222-4246.

Diego-McGlone, M.L.S., Azanza, R.V., Villanoy, C.L., Jacinto, G.S., 2008. Eutrophic waters, algal bloom and fish kill in fish farming areas in Bolinao, Pangasinan, Philippines. Marine Pollution Bulletin, 57, 295-301.

Mhlanga, L., Day, J., Chimbari, M., Siziba, N., Cronberg, G., 2006. Observations on limnological conditions associated with a fish kill of Oreochromis niloticus in Lake Chivero following collapse of an algal bloom. African Journal of Ecology, 44, 199-208.

Nishizawa, T., Asayama, M., Shirai, M., 2001. Cyclic heptapeptide microcystin biosynthesis requires the glutamate racemase gene. Microbiology-Sgm, 147, 1235-1241.

Steiner, C., Glaser, B., Teixeira, W.G., Lehmann, J., Blum, W.E.H., Zech, W., 2008. Nitrogen retention and plant uptake on a highly weathered central Amazonian Ferralsol amended with compost and charcoal. Journal of Plant Nutrition and Soil Science-Zeitschrift Fur Pflanzenernahrung Und Bodenkunde, 171, 893-899.

van den Hoek, C., Mann, D.G., Jahns, H.M., 1995. Introduction to Phycology. Cambridge University Press, New York, NY.